Ultrastructural Analysis of the Large Neuronal Perikarya in an Injured Dentate Nucleus Using an Experimental Model of Hyperthermia-Induced Convulsions: The First Qualitative and Quantitative Study.

Background: Febrile seizures are a common form of convulsions in childhood, with poorly known cellular mechanisms. The objective of this pioneering study was to provide qualitative and quantitative ultrastructural research on the large neuronal perikarya in the cerebellar dentate nucleus (DN), using an experimental model of hyperthermia-induced seizures (HSs), comparable to febrile seizures in children. Methods: The study used young male Wistar rats, divided into experimental and control groups. The HSs were evoked by a hyperthermic water bath at 45 °C for 4 min for four consecutive days. Specimens (1 mm3) collected from the DN were routinely processed for transmission electron microscopy studies. Results: The ultrastructure of the large neurons in the DN affected by hyperthermic stress showed variously pronounced lesions in the perikarya, including total cell disintegration. The most pronounced neuronal lesions exhibited specific morphological signs of aponecrosis, i.e., dark cell degeneration ('dark neurons'). In close vicinity to the 'dark neurons', the aponecrotic bodies were found. The findings of this qualitative ultrastructural study correspond with the results of the morphometric analysis of the neuronal perikarya. Conclusions: Our results may constitute interesting comparative material for similar submicroscopic observations on large DN neurons in HS morphogenesis and, in the future, may help to find potential treatment targets to prevent febrile seizures or reduce recurrent seizures in children.

Selection of X-ray Tube Settings for Relative Bone Density Quantification in the Knee Joint of Cats Using Computed Digital Absorptiometry.

Bone mineral density (BMD) varies with aging and both systemic and local diseases; however, such evidence is lacking in feline medicine. This may be due to the need for general anesthesia in cats for direct BMD measurements using dual-energy X-ray absorptiometry (DXA) or quantitative computed tomography (QCT). In this study, computed digital absorptiometry (CDA), an indirect relative BMD-measuring method, was optimized to select an X-ray tube setting for the quantitative assessment of the feline knee joint. The knee joints of nine cats were radiographically imaged and processed using the CDA method with an aluminum density standard and five X-ray tube settings (from 50 to 80 kV; between 1.2 and 12 mAs). The reference attenuation of the X-ray beam for ten steps (S1-S10) of the density standard was recorded in Hounsfield units (HU), compared between X-ray tube settings, and used to determine the ranges of relative density applied for radiograph decomposition. The relative density decreased (p < 0.0001) with an increase in kV and dispersed with an increase in mAs. Then, the percentage of color pixels (%color pixels), representing ranges of relative density, was compared among S1-S10 and used for the recognition of background artifacts. The %color pixels was the highest for low steps and the lowest for high steps (p < 0.0001), regardless of X-ray tube settings. The X-ray tube setting was considered the most beneficial when it effectively covered the lowest possible HU ranges without inducing background artifacts. In conclusion, for further clinical application of the CDA method for quantitative research on knee joint OA in cats, 60 kV and 1.2 mAs settings are recommended.

Relationship between Feline Knee Joint Osteoarthritis and Bone Mineral Density Quantified Using Computed Tomography and Computed Digital Absorptiometry.

Osteoarthritis (OA), including knee joint OA, is a common chronic condition in cats. In both cats and humans, knee joint OA is characterized radiographically by the presence of osteophytes, enthesiophytes, subchondral sclerosis, and joint space narrowing. However, only in humans have these radiographic signs been reported to increase bone mineral density (BMD). Therefore, this study aims to quantify the volumetric (vBMD) and relative (rBMD) BMD measures of the feline knee joint and compare BMD measures between various severities of OA to test the hypothesized OA-BMD relationship in the knee joint in cats. The 46 feline knee joints were imaged using computed tomography (CT) and conventional radiography supported by the computed digital absorptiometry (CDA) method to obtain vBMD and rBMD, respectively. Both BMD measures were assessed in three regions of interest (ROIs): the distal femur (ROI 1), patella (ROI 2), and proximal tibia (ROI 3). In all locations, vBMD and rBMD showed moderate (ROI 2: r = 0.67, p < 0.0001) to strong (ROI 1: ρ = 0.96, p < 0.0001; ROI 3: r = 0.89, p < 0.0001) positive correlations. Due to differences (p < 0.0001) in the width of the distal femur (17.9 ± 1.21 mm), patella (8.2 ± 0.82 mm), and proximal tibia (19.3 ± 1.16 mm), the rBMD was corrected (corr rBMD) using the thickness coefficient of 0.46 ± 0.04 for ROI 2 and 1.08 ± 0.03 for ROI 3. Regardless of the quantification method used, BMD measures increased linearly from a normal knee joint to severe OA, with differences in BMD between normal and mild to severe knee joint OA. The OA-BMD relationship in the feline knee joint can be preliminarily confirmed.

De novo design of miniprotein antagonists of cytokine storm inducers.

Cytokine release syndrome (CRS), commonly known as cytokine storm, is an acute systemic inflammatory response that is a significant global health threat. Interleukin-6 (IL-6) and interleukin-1 (IL-1) are key pro-inflammatory cytokines involved in CRS and are hence critical therapeutic targets. Current antagonists, such as tocilizumab and anakinra, target IL-6R/IL-1R but have limitations due to their long half-life and systemic anti-inflammatory effects, making them less suitable for acute or localized treatments. Here we present the de novo design of small protein antagonists that prevent IL-1 and IL-6 from interacting with their receptors to activate signaling. The designed proteins bind to the IL-6R, GP130 (an IL-6 co-receptor), and IL-1R1 receptor subunits with binding affinities in the picomolar to low-nanomolar range. X-ray crystallography studies reveal that the structures of these antagonists closely match their computational design models. In a human cardiac organoid disease model, the IL-1R antagonists demonstrated protective effects against inflammation and cardiac damage induced by IL-1ß. These minibinders show promise for administration via subcutaneous injection or intranasal/inhaled routes to mitigate acute cytokine storm effects.

Enhancing the porosity of chitosan sponges with CBD by adding antimicrobial violacein.

Given the growing interest in non-toxic materials with good anti-inflammatory and antimicrobial mechanical properties, this work focuses on preparing chitosan sponges with violacein and cannabis oil crosslinked with dialdehyde chitosan. The sponge was tested for its physicochemical and biological properties, presenting a high swelling rate, good thermal stability, and satisfactory mechanical properties. The obtained sponge's water vapor transmission rate was 2101 g/m2/day and is within the recommended values for ideal wound dressings. Notably, adding violacein favorably affected the material's porosity, which is essential for dressing materials. In addition, studies have shown that the designed material interacts with human serum albumin and exhibits good antioxidant and anti-inflammatory properties. The antibacterial properties of the prepared biomaterial were assessed using the Microtox test against A. fisherii (Gram-negative bacterium) and S. aureus (Gram-positive bacterium). The investigated material provides potential therapeutic benefits due to the synergistic action of chitosan, violacein, and cannabis oil so that it could be used as a dressing material. The natural origin of the substances could provide an attractive and sustainable alternative to traditional commercially available dressings.

Three-Dimensional Segmentation of Equine Paranasal Sinuses in Multidetector Computed Tomography Datasets: Preliminary Morphometric Assessment Assisted with Clustering Analysis.

The paranasal sinuses, a bilaterally symmetrical system of eight air-filled cavities, represent one of the most complex parts of the equine body. This study aimed to extract morphometric measures from computed tomography (CT) images of the equine head and to implement a clustering analysis for the computer-aided identification of age-related variations. Heads of 18 cadaver horses, aged 2-25 years, were CT-imaged and segmented to extract their volume, surface area, and relative density from the frontal sinus (FS), dorsal conchal sinus (DCS), ventral conchal sinus (VCS), rostral maxillary sinus (RMS), caudal maxillary sinus (CMS), sphenoid sinus (SS), palatine sinus (PS), and middle conchal sinus (MCS). Data were grouped into young, middle-aged, and old horse groups and clustered using the K-means clustering algorithm. Morphometric measurements varied according to the sinus position and age of the horses but not the body side. The volume and surface area of the VCS, RMS, and CMS increased with the age of the horses. With accuracy values of 0.72 for RMS, 0.67 for CMS, and 0.31 for VCS, the possibility of the age-related clustering of CT-based 3D images of equine paranasal sinuses was confirmed for RMS and CMS but disproved for VCS.

Structure of the interleukin-5 receptor complex exemplifies the organizing principle of common beta cytokine signaling.

Cytokines regulate immune responses by binding to cell surface receptors, including the common subunit beta (ßc), which mediates signaling for GM-CSF, IL-3, and IL-5. Despite known roles in inflammation, the structural basis of IL-5 receptor activation remains unclear. We present the cryo-EM structure of the human IL-5 ternary receptor complex, revealing architectural principles for IL-5, GM-CSF, and IL-3. In mammalian cell culture, single-molecule imaging confirms hexameric IL-5 complex formation on cell surfaces. Engineered chimeric receptors show that IL-5 signaling, as well as IL-3 and GM-CSF, can occur through receptor heterodimerization, obviating the need for higher-order assemblies of ßc dimers. These findings provide insights into IL-5 and ßc receptor family signaling mechanisms, aiding in the development of therapies for diseases involving deranged ßc signaling.

Influence of the Type of Biocompatible Polymer in the Shell of Magnetite Nanoparticles on Their Interaction with DPPC in Two-Component Langmuir Monolayers.

Magnetite nanoparticles (MNPs) are attractive nanomaterials for applications in magnetic resonance imaging, targeted drug delivery, and anticancer therapy due to their unique properties such as nontoxicity, wide chemical affinity, and intrinsic superparamagnetism. Their functionalization with polymers such as chitosan or poly(vinyl alcohol) (PVA) can not only improve their biocompatibility and biodegradability but it also plays an important role in their interactions with biological cells. In this work, the effect of the functionalization of MNPs with chitosan, PVA, and their blend on model cell membranes formed from 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC) using a Langmuir technique was studied. The studies performed showed that the type of biocompatible polymer in the MNP shell plays a crucial role in the effectiveness of its adsorption process into the model cell membrane. Modification of MNPs with chitosan facilitates significantly more effective adsorption than coating them with PVA or with a chitosan and PVA blend. The presence of all the investigated MNPs in the DPPC monolayer at low concentrations does not affect its thermodynamic state, fluidity, or morphology, which is promising in terms of their biocompatibility. On the other hand, their high concentration (molar fraction above ≈0.05) exerts a disruptive effect on the model cell membrane and results in their aggregation, leading probably to the loss of their superparamagnetic properties essential for nanomedicine.

Photochemical stability of chitosan films doped with cannabis oil.

The effect of UV radiation from three different sources on chitosan (CS) films containing the addition of 10% by weight of cannabis oil was investigated. Cannabis oil (CBD) alone exposed to UV is unstable, but its photostability significantly increases in the chitosan matrix. The course of photochemical reactions, studied by FTIR spectroscopy, is slow and inefficient in chitosan with CBD, even under high-energy UV sources. The research also included chitosan films with CBD cross-linked with dialdehyde starch (DAS). Using AFM microscopy and contact angle measurements, the morphology and surface properties of prepared chitosan films with CBD were investigated, respectively. It was found that CBD embedded in CS is characterized by the best photostability under the influence of an LED emitting long-wave radiation. Using a monochromatic and polychromatic UV lamp (HPK and UV-C) emitting high-energy radiation, gradual degradation accompanied by oxidation was observed, both in the CS chains and in the CBD additive. Additionally, changes in surface properties are observed during UV irradiation. It was concluded that CS protects CBD against photodegradation, and a further improvement in photochemical stability is achieved after system cross-linking with DAS.

Three-Dimensional Segmentation Assisted with Clustering Analysis for Surface and Volume Measurements of Equine Incisor in Multidetector Computed Tomography Data Sets.

Dental diagnostic imaging has progressed towards the use of advanced technologies such as 3D image processing. Since multidetector computed tomography (CT) is widely available in equine clinics, CT-based anatomical 3D models, segmentations, and measurements have become clinically applicable. This study aimed to use a 3D segmentation of CT images and volumetric measurements to investigate differences in the surface area and volume of equine incisors. The 3D Slicer was used to segment single incisors of 50 horses' heads and to extract volumetric features. Axial vertical symmetry, but not horizontal, of the incisors was evidenced. The surface area and volume differed significantly between temporary and permanent incisors, allowing for easy eruption-related clustering of the CT-based 3D images with an accuracy of >0.75. The volumetric features differed partially between center, intermediate, and corner incisors, allowing for moderate location-related clustering with an accuracy of >0.69. The volumetric features of mandibular incisors' equine odontoclastic tooth resorption and hypercementosis (EOTRH) degrees were more than those for maxillary incisors; thus, the accuracy of EOTRH degree-related clustering was >0.72 for the mandibula and >0.33 for the maxilla. The CT-based 3D images of equine incisors can be successfully segmented using the routinely achieved multidetector CT data sets and the proposed data-processing approaches.

Polymer-Based Nanoparticles as Drug Delivery Systems for Purines of Established Importance in Medicine.

Many purine derivatives are active pharmaceutical ingredients of significant importance in the therapy of autoimmune diseases, cancers, and viral infections. In many cases, their medical use is limited due to unfavorable physicochemical and pharmacokinetic properties. These problems can be overcome by the preparation of the prodrugs of purines or by combining these compounds with nanoparticles. Herein, we aim to review the scientific progress and perspectives for polymer-based nanoparticles as drug delivery systems for purines. Polymeric nanoparticles turned out to have the potential to augment antiviral and antiproliferative effects of purine derivatives by specific binding to receptors (ASGR1-liver, macrophage mannose receptor), increase in drug retention (in eye, intestines, and vagina), and permeation (intranasal to brain delivery, PEPT1 transport of acyclovir). The most significant achievements of polymer-based nanoparticles as drug delivery systems for purines were found for tenofovir disoproxil in protection against HIV, for acyclovir against HSV, for 6-mercaptopurine in prolongation of mice ALL model life, as well as for 6-thioguanine for increased efficacy of adoptively transferred T cells. Moreover, nanocarriers were able to diminish the toxic effects of acyclovir, didanosine, cladribine, tenofovir, 6-mercaptopurine, and 6-thioguanine.

Biochemical and biophysical characterization of natural polyreactivity in antibodies.

To become specialized binders, antibodies undergo a process called affinity maturation to maximize their binding affinity. Despite this process, some antibodies retain low-affinity binding to diverse epitopes in a phenomenon called polyreactivity. Here we seek to understand the molecular basis of this polyreactivity in antibodies. Our results highlight that polyreactive antigen-binding fragments (Fabs) bind their targets with low affinities, comparable to T cell receptor recognition of autologous classical major histocompatibility complex. Extensive mutagenic studies find no singular amino acid residue or biochemical property responsible for polyreactive interaction, suggesting that polyreactive antibodies use multiple strategies for engagement. Finally, our crystal structures and all-atom molecular dynamics simulations of polyreactive Fabs show increased rigidity compared to their monoreactive relatives, forming a neutral and accessible platform for diverse antigens to bind. Together, these data support a cooperative strategy of rigid neutrality in establishing the polyreactive status of an antibody molecule.

Fabrication and characterization of new levan@CBD biocomposite sponges as potential materials in natural, non-toxic wound dressing applications.

Wound healing is a complex process; therefore, new dressings are frequently required to facilitate it. In this study, porous bacterial levan-based sponges containing cannabis oil (Lev@CBDs) were prepared and fully characterized. The sponges exhibited a suitable swelling ratio, proper water vapor transmission rate, sufficient thermal stability, desired mechanical properties, and good antioxidant and anti-inflammatory properties. The obtained Lev@CBD materials were evaluated in terms of their interaction with proteins, human serum albumin and fibrinogen, of which fibrinogen revealed the highest binding effect. Moreover, the obtained biomaterials exhibited antibacterial activity against Staphylococcus aureus and Pseudomonas aeruginosa, as well as being non-hemolytic material as indicated by hemolysis tests. Furthermore, the sponges were non-toxic and compatible with L929 mouse fibroblasts and HDF cells. Most significantly, the levan sponge with the highest content of cannabis oil, in comparison to others, retained its non-hemolytic, anti-inflammatory, and antimicrobial properties after prolonged storage in a climate chamber at a constant temperature and relative humidity. The designed sponges have conclusively proven their beneficial physicochemical properties and, at the preliminary stage, biocompatibility as well, and therefore can be considered a promising material for wound dressings in future in vivo applications.

An application of the density standard and scaled-pixel-counting protocol to assess the radiodensity of equine incisor teeth affected by resorption and hypercementosis: preliminary advancement in dental radiography.

BACKGROUND: Equine Odontoclastic Tooth Resorption and Hypercementosis (EOTRH) syndrome is a dental disease where the radiographic signs may be quantified using radiographic texture features. This study aimed to implement the scaled-pixel-counting protocol to quantify and compare the image structure of teeth and the density standard in order to improve the identification of the radiographic signs of tooth resorption and hypercementosis using the EOTRH syndrome model. METHODS AND RESULTS: A detailed examination of the oral cavity was performed in 80 horses and maxillary incisor teeth were evaluated radiographically, including an assessment of the density standard. On each of the radiographs, pixel brightness (PB) was extracted for each of the ten steps of the density standard (S1-S10). Then, each evaluated incisor tooth was assigned to one of 0-3 EOTRH grade-related groups and annotated using region of interest (ROI). For each ROI, the number of pixels (NP) from each range was calculated. The linear relation between an original X-ray beam attenuation and PB was confirmed for the density standard. The NP values increased with the number of steps of the density standard as well as with EOTRH degrees. Similar accuracy of the EOTRH grade differentiation was noted for data pairs EOTRH 0-3 and EOTRH 0-1, allowing for the differentiation of both late and early radiographic signs of EOTRH. CONCLUSION: The scaled-pixel-counting protocol based on the use of density standard has been successfully implemented for the differentiation of radiographic signs of EOTRH degrees.

Bio-Approach for Obtaining Enantiomerically Pure Clopidogrel with the Use of Ionic Liquids.

Clopidogrel is a chiral compound widely used as an antiplatelet medication that lowers the risk of blood clots, strokes, and heart attacks. The main aim of the study presented herein was to obtain (S)-clopidogrel, which is commercially available in treatments, via the kinetic resolution of racemic clopidogrel carboxylic acid with the use of lipase from Candida rugosa and a two-phase reaction medium containing an ionic liquid. For this purpose, the enantioselective biotransformation of clopidogrel carboxylic acid and chiral chromatographic separation with the use of a UPLC-MS/MS system were optimized. The best kinetic resolution parameters were obtained by using a catalytic system containing lipase from Candida rugosa OF as a biocatalyst, cyclohexane and [EMIM][BF4] as a two-phase reaction medium, and methanol as an acyl acceptor. The enantiomeric excess of the product was eep = 94.21% ± 1.07 and the conversion was c = 49.60% ± 0.57%, whereas the enantioselectivity was E = 113.40 ± 1.29. The performed study proved the possibility of obtaining (S)-clopidogrel with the use of lipase as a biocatalyst and a two-phase reaction medium containing an ionic liquid, which is in parallel with green chemistry methodology and does not require environmentally harmful conditions.

Correction: Górski et al. Application of Two-Dimensional Entropy Measures to Detect the Radiographic Signs of Tooth Resorption and Hypercementosis in an Equine Model. Biomedicines 2022, 10, 2914.

In the original article [...].

Chitosan Composites Containing Boron-Dipyrromethene Derivatives for Biomedical Applications.

The work is devoted to preparing and characterizing the properties of photosensitive composites, based on chitosan proposed for photodynamic therapy. Chitosan films with a 5% addition of two BODIPY dyes were prepared by solution casting. These dyes are dipyrromethene boron derivatives with N-alkyl phthalimide substituent, differing in the presence of iodine atoms in positions 2 and 6 of the BODIPY core. The spectral properties of the obtained materials have been studied by infrared and UV-vis absorption spectroscopy and fluorescence, both in solutions and in a solid state. Surface properties were investigated using the contact angle measurement. The morphology of the sample has been characterized by Scanning Electron and Atomic Force Microscopy. Particular attention was paid to studying the protein absorption and kinetics of the dye release from the chitosan. Adding BODIPY to the chitosan matrix leads to a slight increase in hydrophilicity, higher structure heterogeneity, and roughness, than pure chitosan. The presence of iodine atoms in the BODIPY structure caused the bathochromic effect, but the emission quantum yield decreased in the composites. It has been found that BODIPY-doped chitosan interacts better with human serum albumin and acidic α-glycoprotein than unmodified chitosan. The release rate of dyes from films immersed in methanol depends on the iodine present in the structure.

Ensemble of Heterogeneous Base Classifiers for Human Gait Recognition.

Human gait recognition is one of the most interesting issues within the subject of behavioral biometrics. The most significant problems connected with the practical application of biometric systems include their accuracy as well as the speed at which they operate, understood both as the time needed to recognize a particular person as well as the time necessary to create and train a biometric system. The present study made use of an ensemble of heterogeneous base classifiers to address these issues. A Heterogeneous ensemble is a group of classification models trained using various algorithms and combined to output an effective recognition A group of parameters identified on the basis of ground reaction forces was accepted as input signals. The proposed solution was tested on a sample of 322 people (5980 gait cycles). Results concerning the accuracy of recognition (meaning the Correct Classification Rate quality at 99.65%), as well as operation time (meaning the time of model construction at <12.5 min and the time needed to recognize a person at <0.1 s), should be considered as very good and exceed in quality other methods so far described in the literature.

Facile repurposing of peptide-MHC-restricted antibodies for cancer immunotherapy.

Monoclonal antibodies (Abs) that recognize major histocompatability complex (MHC)-presented tumor antigens in a manner similar to T cell receptors (TCRs) have great potential as cancer immunotherapeutics. However, isolation of 'TCR-mimic' (TCRm) Abs is laborious because Abs have not evolved the structurally nuanced peptide-MHC restriction of αß-TCRs. Here, we present a strategy for rapid isolation of highly peptide-specific and 'MHC-restricted' Abs by re-engineering preselected Abs that engage peptide-MHC in a manner structurally similar to that of conventional αß-TCRs. We created structure-based libraries focused on the peptide-interacting residues of TCRm Ab complementarity-determining region (CDR) loops, and rapidly generated MHC-restricted Abs to both mouse and human tumor antigens that specifically killed target cells when formatted as IgG, bispecific T cell engager (BiTE) and chimeric antigen receptor-T (CAR-T). Crystallographic analysis of one selected pMHC-restricted Ab revealed highly peptide-specific recognition, validating the engineering strategy. This approach can yield tumor antigen-specific antibodies in several weeks, potentially enabling rapid clinical translation.

The Postural and Body Surface Temperature Response of Leisure Horses to Lunging with Selected Lunging Aids.

Incorporating lunging into a horse's daily routine aims to enhance fitness, physical condition, and specific skills or exercises when using lunging aids (LAs). To assess the effectiveness of lunging, non-contact technologies like geometric morphometrics and infrared thermography can be employed. This study seeks to evaluate lunging efficiency based on the horse's posture and surface temperature when lunging with different head and neck positions. The study aims to determine if changes in a horse's posture correspond to increased metabolic activity, as indicated by body surface temperature. Thirteen horses included in the study were lunged with chambon (CH), rubber band (RB), and triangle side reins (TRs) as well as with a freely moving head (FMH). Images were taken in visible light and infrared. Principal Component Analysis (PCA) was used to analyze horse posture changes and a Pixel-Counting Protocol (PCP) was used to quantify surface temperature patterns. The horses' posture exhibited contrasting changes, reflected by a changing centroid shape (p < 0.0001) but not size (p > 0.05) when lunged with RB and TRs, but not CH. Different (p < 0.0001) surface temperature patterns were observed during lunging. FMH lunging resulted in lower temperatures over a larger surface, CH induced moderate temperatures on a smaller area, RB caused moderate to high temperatures across a broader surface, and TRs led to higher temperatures over a smaller region. The studied lunging cases returned different (p < 0.0001) surface temperature patterns. Lunging with FMH returned lower temperatures over a larger surface, CH moderate temperatures on a smaller area, RB moderate to high temperatures across a broader surface, and TRs higher temperatures over a smaller region. The proposed methods can be applied to evaluate the efficiency of lunging in horses.